The majority of retroviral vectors which are presently used in gene therapeutic research are derived from the amphotropic murine leukemia virus (MLV). The host cell range of the amphotropic MLV is determined by the surface envelope protein (SU) encoded by the env gene. The protein products of the env gene form the outer envelope of the retroviral vector. The SU proteins interact with, i.e. bind to a specific protein (receptor) on the surface of the host cell. The env gene products of the amphotropic MLV enable gene transfer into a great number of different mammalian cells. However, a selective gene transfer in particular cell or tissue types of humans or other mammals is not possible, since the receptor for the MLV envelope proteins on the surface of mammalian cells, which mediates the entry of amphotropic MLV vectors and gene transfer, is found on nearly all of these cells. Accordingly, the host cell range of the amphotropic MLV is not specific.
A host cell specificity, however, is advantageous e.g. for gene therapeutic use, since in a gene therapy outside of the organism (ex vivo) (Anderson et al., Science 256 (1992) 808-813; Yu et al., H. Gene Therapy 8 (1997) 1065-1072) laborious purifications of cells are avoided. It is desired for therapeutic, diagnostic or vaccination use in vivo, that retroviral vectors are targeted specifically to the desired host cells and subsequently transfer the therapeutic gene. By modification of the surface envelope protein a restriction of the host cell range of the amphotropic MLV could be achieved. A modification of the surface capsid protein was done by fusion with a hormone domain. A transduction of the cells carrying the specific hormone receptor occurred (Kasahara et al., Science 266 (1994) 1373-1375). Further, the surface envelope protein was modified by fusion with a single chain antibody fragment (single chain variable fragment, in the following referred to as “scFv”). The fragment represented the antigen binding domain of an antibody and is a fusion protein composed of the variable domains Vh and Vl of a monoclonal antibody. Both domains are linked via a glycine and serine oligopeptide [-(ser-gly4)3-gly-)] which enables the correct folding of the fusion protein (Huston et al., Methods Enzymol. 203 (1991) 46-88, Whitlow et al., Methods: A companion to Methods Enzymol. 2 (1991) 97-105). All modifications of the MLV surface capsid protein using a scFv carried out so far showed that while binding of the vectors to the host target cell occurred, however, there was no entry into the cell (Russel et al., Nucleic Acid Res. 21 (1993) 1081-1085). Furthermore it is known that the surface envelope protein of the MLV generally does not enable for extensive modifications (Cosset et al., J. Virol. 69 (1995) 6314-632). Modifications in which a portion of the binding domain of the MLV-SU protein has been replaced led to an incorrect processing and, thus, to a defective transport of the SU protein to the cell surface (Weiss et al., In J. A. Levy (ed.) The Retroviridae 2 (1993) 1-108; Morgan et al., J. Virol. 67 (1993) 4712-4721; Russel et al., Nucleic Acid Res. 21 (1993) 1081-1085). Accordingly, the development of cell-specific retroviral vectors on the basis of MLV with modified surface envelope proteins is only little promising.
Retroviral vectors on the basis of spleen necrosis virus SNV are more suitable for a targeted gene transfer into e.g. human cells, since the surface envelope protein of SNV enables for extensive modifications after which still correct processing occurs (Martinez and Dornburg, Virol. 208 (1995) 234-241; Chu et al., Gene Therapy 1 (1994) 292-299; Chu and Dornburg J. Virol. 69 (1995) 2659-2663). At least two components are required for the preparation of such vectors. On the one hand a so-called expression construct is to be prepared enabling packaging in and transfer through a retrovirus. The expression construct comprises a coding DNA fragment of the desired gene product, e.g. a gene for gene therapy or as a vaccine. The expression construct has to comprise a nucleotide sequence which is referred to as packaging signal psi (ψ) and controls the efficient packaging of mRNA in retroviral particles. Furthermore a packaging or helper cell is required which provides the gag, pol and env gene products of SNV, without packaging of the gag, pol and env genes into a retrovirus. The gag, pol and env genes which are present in the packaging cell have to be psi-negative. Following transduction of the expression construct by means of transfection of the respective plasmid DNA into the packaging cells, retroviral particles are released into the cell culture supernatant, which particles contain the expression construct and are only able to transduct said construct but not the gag, pol and env genes into the target cell. Said vectors are replication incompetent and only pass one cycle of replication. The general method for the preparation of replication incompetent retroviral vectors is known in the prior art (Weiss et al., In J. A. Levy (ed.). The Retroviridae 2 (1993) 1-108; Morgan et al., J. Virol. 67 (1993) 4712-4721; Russel et al., Nucleic Acid Res. 21 (1993) 1081-1085; Cosset et al., J. Virol 69 (1995); Martinez and Dornburg, Virol. 208 (1995) 234-241; Chu et al., Gene Therapy 1 (1994) 292-299; Chu and Dornburg, J. Virol. 69 (1995) 2659-2663).
The tropism (host cell specificity) of spleen necrosis virus is determined by the surface envelope protein (SU protein) encoded by the env gene of SNV. The wild type SNV surface envelope protein does not allow for a selective gene transfer into particular human cells or tissues since the specific acceptor protein (receptor) is not present on the surface of human cells (Dornburg, Gene Therapy 2 (1995) 1-10). Therefore, a method has been developed to substitute the SU protein of SNV by the antigen recognizing domains of antibodies. Said [SNV-scFv-Env] vectors with the two different scFv known heretofore were able to transmit the psi positive reporter gene, bacterial β-galactosidase, into the selected human target cells. Said scFv are directed against the hapten dinitrophenol (DNP) or against an unknown surface molecule on colon CA cells and other cancer cells, respectively. (Chu et al., Gene Therapy 1 (1994) 292-299, Chu et al., BioTechniques 18 (1995) 890-899; Chu and Dornburg, J. Virol. 71 (1997) 720-725). A packaging cell line (DSH-CXL) has been developed, containing the psi-negative SNV genes gag, pol and env as well as the psi-positive reporter gene expression construct (pCXL). Following transfection of the packaging cell using plasmid DNA of another env expression gene (pTC53), in which the entire surface envelope protein was substituted by a single chain antibody fragment (scFv), retroviral vectors were released into the cell supernatant which in addition to the wild type surface envelope protein also carried the chimeric [scFv-Env] surface protein on their surface. By means of said vectors the reporter gene could be transferred into the target cells specific for scFv, canine osteosarcoma cells (D17), which were conjugated with DNP, or HeLA cells (human cervical carcinoma cells), respectively. However, this method described for the preparation of cell-specific retroviral vectors has the disadvantage that only already known and cloned scFv may be used. Further, it has been found by us that not every scFv is suitable as a portion of a [SNV-scFv-Env] vector for cell transduction (transfer of the desired gene to the target cell).